Touch imprint cytology with massively parallel sequencing (TIC-seq): a simple and rapid method to snapshot genetic alterations in tumors.
Author(s): Amemiya K, Hirotsu Y, Goto T, Nakagomi H, Mochizuki H, Oyama T, Omata M
Publication: Cancer Med, 2016, Vol. 5, Page 3426-3436
PubMed ID: 27774772 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if touch imprint cytology (TIC) tumor specimens (that were air-dried and Giemsa-stained or ethanol-fixed and Pap-stained) are an adequate substitute for formalin-fixed, paraffin-embedded (FFPE) specimens for the detection of somatic mutations by targeted sequencing in lung and breast cancer.
Conclusion of Paper
On average, fewer slides were required to extract Sufficient DNA for sequencing from TIC specimens than FFPE specimens (1 versus 3.4 slides). The mean amount of long DNA (268 bp amplicon of the RNase P gene) isolated from a slide was greatest for TIC specimens that were Giesma-stained (1009.3 ng), followed by those that were Pap-stained (593.2 ng), and FFPE specimens that were H&E-stained (90.4 ng). DNA isolated from Giesma-stained TIC specimens had significantly higher relative quantitative (RQ) values (ratio of long (289 bp) to short (87 bp) amplicons of the RNase P gene) (mean=~0.82) than Pap-stained TIC specimens (mean=~0.55, P=0.04) and H&E-stained FFPE specimens (mean=~0.42, P=0.002). Sequencing quality metric data were comparable among the three specimen types, with similar numbers of mapped reads, percent on target reads, mean read lengths, and base call accuracy. The average depth of sequencing coverage was also similar among DNA from Giesma-stained TIC specimens (864×), Pap-stained TIC specimens (717×), and H&E-stained FFPE specimens (717×). Of the 79 somatic mutations detected, 72 were consistent across the three specimen types (92% concordance); concordance was 100% for EGFR mutations among the three specimen types from the 6 lung adenocarcinomas evaluated. Immunohistochemistry of a breast cancer specimen revealed that the primary FFPE tumor included two histologically distinct areas, an ER-, PR-, HER2- lesion and an ER+, PR+, HER2- lesion), only one of which was captured by TICseq analysis, indicating that TICseq can discriminate between clonal subpopulations in a heterogenous primary tumor specimen.
Studies
-
Study Purpose
The purpose of this paper was to determine if touch imprint cytology (TIC) tumor specimens are an adequate surrogate for FFPE tumor specimens for the detection of somatic mutations by targeted sequencing in lung and breast cancer. Surgically resected lung cancer specimens (6 adenocarcinomas, 3 squamous cell carcinoma) and one breast cancer specimen (primary tumor) and three lymph nodes (metastatic tumors) were used to collect TIC and FFPE specimens. TIC specimens were collected by touching >80% of a PEN Membrane Glass Slide against the cut edge of the fresh surgically resected tumor. TIC slides were air-dried and Giemsa stained or fixed in 95% ethanol and Pap stained to assess tumor cellularity. FFPE tissue sections (10 µm thick) were slide-mounted and stained with hematoxylin and eosin to determine tumor cellularity. All slides were screened by a cytopathologist and stored at 4°C. If tumor cellularity was <70%, tumor cellularity was enriched by macro- or laser-capture microdissection dissection. DNA was extracted from TIC and FFPE slides with the QIAamp DNA FFPE Kit. DNA integrity was assessed by real-time PCR using the TaqMan RNase P Detection Reagents Kit and the FFPE SNA QC Assay v2, and the extent of genomic DNA fragmentation was estimated by calculating the relative quantification (RQ) value of long (289 bp) to short (87 bp) amplicons of RNase P. Targeted sequencing of DNA from stained TIF and FFPE slides was performed by multiplex PCR with the Ion AmpliSeq Library Kit 2.0 and custom-designed panels targeting 53 genes each for breast and lung cancer. Libraries were sequenced on an Ion PGM System or on an Ion Proton. The ion Torrent Suite Software was used to process sequencing data.
Summary of Findings:
On average, fewer slides were required to extract sufficient DNA for sequencing from TIC specimens than FFPE specimens (1 versus 3.4 slides). The mean amount of long DNA (268 bp amplicon of the RNase P gene) isolated from a slide was greatest for TIC specimens that were Giesma-stained (1009.3 ng), followed by those that were Pap-stained (593.2 ng), and FFPE specimens that were H&E-stained (90.4 ng). DNA isolated from Giesma-stained TIC specimens had significantly higher RQ values (ratio of 289 to 87 bp amplicon of RNase P) (mean=~0.82), than Pap-stained TIC specimens ((mean=~0.55, P=0.04) and H&E-stained FFPE specimens (mean=~0.42, P=0.002). Sequencing quality metric data were comparable among the three specimen types, with similar numbers of mapped reads, percent on target reads, mean read lengths, and base call accuracy. The average depth of sequencing coverage was also similar among DNA from Giesma-stained TIC specimens (864×), Pap-stained TIC specimens (717×), and H&E-stained FFPE specimens (717×). Of the 79 somatic mutations detected, 72 were consistent across the three specimen types (92% concordance); concordance was 100% for EGFR mutations among the three specimen types from the 6 lung adenocarcinomas evaluated. Immunohistochemistry of a breast cancer specimen revealed that the primary FFPE tumor included two histologically distinct areas, an ER-, PR-, HER2- lesion and an ER+, PR+, HER2- lesion), only one of which was captured by TICseq analysis, indicating that TICseq can discriminate between clonal subpopulations in a heterogenous primary tumor specimen.
Biospecimens
Preservative Types
- Formalin
- None (Fresh)
- Ethanol
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR Protein Immunohistochemistry DNA DNA sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Ethanol
Air-dried
DNA sequencing Specific Type of tissue stain Giemsa stain
Pap stain
Biospecimen Acquisition Method of cell acquisition Touch imprint cytology
FFPE tissue section